The goal of this renewal proposal is to continue our approach that combines phage antibody display with laser capture micro dissection (LCM) to identify novel internalizing human single chain antibodies (scFvs) that target metastatic tumor cells in situ residing in their natural tissue microenvironment. The work is built on our previous work where we developed and used this novel approach to identify antibodies targeting primary prostate tumor. This proposal aims to advance our study to prostate tumor bone metastases, a major challenge for treatment and management of the disease. This proposal aims to use our novel LCM-based human antibody library selection strategy to identify novel scFvs that target tumor metastases-associated cell surface antigens expressed by tumor cells in situ for imaging and therapy development. In addition, based on novel findings of the previous R01 study where we found that tumor antigens such as ALCAM are actively cleaved in metastatic prostate cancer cells, we will select antibodies that target cell surface neoepitopes on the post-cleavage transmembrane remnants of ALCAM. Specifically, we aim (1) to develop novel human antibodies that target ALCAM post-cleavage transmembrane remnants. We will use these antibodies to study the prevalence of ALCAM transmembrane remnants on prostate tumor bone metastases by immunohistochemistry, and evaluate utility of anti-remnant scFvs in tumor targeting in vivo using SPECT/CT imaging techniques. (2) To identify rapidly internalizing antibodies that are capable of high-level accumulation in target metastatic prostate cancer cells residing in their tissue microenvironment. We propose to use our previously developed and validated novel LCM-based antibody library selection strategies to identify novel antibodies targeting prostate tumor bone metastases with the following properties: (a) binding to tumor cells in situ. (b) Binding to live tumor cells and thus recognizing tumor antigens in their native conformations. (c) rapidly internalizing and accumulating to high levels inside tumor cells. (d) Exhibiting high-level tumor uptake in vivo and minimal uptake in off target tissues in both localized (subcutaneous xenograft) and disseminated prostate tumor models. (3) To identify tumor antigens bound by our novel antibodies. We will screen a large yeast surface displayed cDNA library developed in the lab to identify tumor antigens bound by our novel phage antibodies. In parallel, we will identify tumor antigens by immunoprecipitation and mass spectrometry analysis that allow identification of post- translational modified antigens.